Identification of Epigenetic Biomarkers in HPV-Related Carcinomas

Head and neck squamous cell carcinomas (HNSCC), especially those localised in the oropharynx and oral cavity, have increased significantly in recent decades, with persistent Human papillomavirus (HPV) infection identified as the main aetiological cause. Conventional clinical and pathological evaluation is often insufficient to accurately assess the risk of malignant transformation in precancerous lesions. This limitation often delays the diagnosis of oral cancer, contributing to unfavourable outcomes for many patients. Therefore, the need for correct patient risk stratification and appropriate patient triage has led to the search for new diagnostic and prognostic molecular biomarkers, specifically among the epigenetic modification of DNA methylation. In this context, the PreCursor-M+ kit (Fujirebio, Tokyo, Japan), designed to analyse hypermethylation of the two onco-suppressor genes FAM19A4 and miR124-2 in cervical samples from high-risk HPV-positive women, was used to assess the abnormal methylation level of 111 oral rinse samples. The samples were distinguished according to the histologically diagnosed lesion: oral squamous cell carcinoma (OSCC), potentially malignant lesions (OPMD) and benign lesions (BL). To these, a group of healthy individuals was added (NL). In each group, samples were selected to have approximately equal numbers of HPV-positive and HPV-negative samples. HPV detection was performed using INNO-LiPA® HPV Genotyping Extra II diagnostic kit (Fujirebio, Tokyo, Japan), which allows the identification of thirty-two distinct genotypes, distinguished into twelve low-risk HPVs and twenty high-risk HPVs. Analysis of the results showed how hypermethylation correlated with the severity of the diagnosis. A positive hypermethylation result, as indicated by the kit, was more common in OSCC (p<0.0001), while a negative result was more common in NL and BL (p= 0.0023 and p= 0.0162). HPV positivity was positively correlated with hypermethylation only in OSCCs (32.4%, p= 0.0006), although statistical significance was also maintained in HPV-negative patients (p= 0.0007). Among HPV-positive OSCC, those infected with HPV16 showed a higher methylation level. Furthermore, the methylation levels of the two targets taken individually increased from the NL group to the BL and OPMD groups and finally to OSCCs. This study has shown how assessing hypermethylation of two targets can help diagnose OSCC and identify those patients affected by OPMDs who could be at risk of developing cancer. The relatively small number of patients did not allow for the detection of statistically significant differences in the effect of HPV infection on target methylation levels. It is therefore imperative to increase the sample size to confirm these preliminary results and to outline new significant information.